Preparation, characteristics and cytotoxicity of green synthesized selenium nanoparticles using Paenibacillus motobuensis LY5201 isolated from the local specialty food of longevity area

Selenium is an essential micronutrient element. For the extremely biotoxic of selenite, Selenium nanoparticles (SeNPs) is gaining increasing interest. In this work, a selenium-enriched strain with highly selenite-resistant (up to 173 mmol/L) was isolated from the local specialty food of longevity area and identified as Paenibacillus motobuensis (P. motobuensis) LY5201. Most of the SeNPs were accumulated extracellular. SeNPs were around spherical with a diameter of approximately 100 nm. The X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy showed that the purified SeNPs consisted of selenium and proteins. Our results suggested that P. motobuensis LY5201could be a suitable and robust biocatalyst for SeNPs synthesis. In addition, the cytotoxicity effect and the anti-invasive activity of SeNPs on the HepG2 showed an inhibitory effect on HepG2, indicating that SeNPs could be used as a potential anticancer drug.

www.nature.com/scientificreports/ oxyanions to elemental selenium 12,13 , providing a simple and environment-friendly method to prepare SeNPs. However, the selenite tolerance of most reported SeNPs producing microbes is relatively low (< 100 mmol/L) 14,15 , and the time for the reduction of these toxic forms is long, ranging from 24 to 96 h 16 . Thus, the identification of novel strains with a high selenium tolerance is urgently needed. In this study, a new Se-reducing bacteria isolate (LY5201) showing tolerance to selenite (173 mmol/L, 30 g/L) was isolated from Chinese Sauerkraut, in Bama, "the hometown of longevity" in the world. We found that SeNPs could be synthesized by Paenibacillus. motobuensis (P. motobuensis) LY5201 efficiently under sodium selenite (Na 2 SeO 3 ) stress in anaerobic conditions. SeNPs could be detected within 24 h, which is faster than reported microbes, indicating that this strain is suitable for SeNPs preparation. The SeNPs characteristics were determined by transmission electron microscopy (TEM) and fourier-transform infrared (FTIR) spectroscopy. Hereafter, the cytotoxicity of SeNPs was investigated. The SeNPs synthesized by P. motobuensis LY5201 may be used as a promising drug for anticancer.

Materials and methods
Selenite-reducing strain isolation and identification. Samples were isolated from Chinese Sauerkraut (Bama, Guangxi, China). The supernatant of the Chinese Sauerkraut was plated on Luria Bertani (LB) agar containing 500 mg/L sodium selenite. After incubated at 37 °C for 24 h, individual colonies are red, indicating Se reduction and Se 0 formation. The single colony was confirmed by 16S rRNA gene sequencing analysis 17 . Isolate 201 (named LY5201) was selected for further study for its rapid growth rate and Se reduction performance. The 16S rRNA gene was amplified and sequenced as previously study 18 . The 16S rRNA gene sequence was compared to previously published sequences present on the EzBioCloud server 19 . A phylogenetic tree was constructed using the maximum likelihood algorithms of MEGA 7 20 .
Assessment of sensitivity of LY5201 to Na 2 SeO 3 . The influence of SeNPs on the proliferation ability of LY5201 was investigated. First, a fresh overnight cell culture without Na 2 SeO 3 was used as seed. Different concentrations of Na 2 SeO 3 (0, 0.5 g/L, 1.0 g/L, 2.0 g/L, 5.0 g/L, 10.0 g/L and 30.0 g/L) were prepared in LB media. The seed was added into these LB media at an initial cell density of 0.3 (OD 600  Characterizations of SeNPs. In order to obtain SeNPs, the sterilized sodium selenite solution was added to the fermentation medium, bringing the final concentration of the solution to 5.0 g/L. The fermentation broth was centrifuged at 15,000 × g for 10 min. The resultant pellet was washed three times and resuspended in 20 mL deionized water. To separate the SeNPs from the cell fragments, 5 mL of 1-octanol was added. The mixture was mixed thoroughly and centrifuged at 2,000 × g for 5 min. Then, the mixture was placed at 4 °C for 24 h. The lower layer containing SeNPs was collected and cleaned consecutively with 70% ethanol, chloroform and deionized sterile water and freeze-dried 21 . To further characterize the SeNPs, transmission electron microscopy (TEM, JEOL-7100), X-ray photoelectron spectroscopy (XPS, Escalab 250Xi), fourier transform infrared spectroscopy (FTIR, 640-IR), dynamic light scattering (DLS, Litesizer 500) and UV-visible analysis (UV-Vis, Varian Cary 100) were carried out as previously described 13 .
Cytotoxicity analysis of SeNPs in vitro. Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China) was used for in vitro cytotoxicity testing of SeNPs. Hepatocarcinoma (HepG2) purchased from the Cell Bank of Chinese academy of Sciences (Shanghai, China) was cultured in DMEM supplemented with 10% fetal bovine serum in 96-well plates. In this experiment, 96 well plates (200 μL media per well) were seeded with cells at final concentration of 1 × 10 5 cells/mL and incubated in 5% CO 2 incubator at 37 °C. After 24 h incubation, SeNPs were added into the culture to keep the final concentrations at 5, 10, 15 and 20 μg/mL The culture was wells incubated in 5% CO 2 incubator at 37 °C for 24 h. Then, each well was treated with 10 μL of CCK-8 and incubated at the same previous condition for 2 h. The color intensity of the solution was measured at 450 nm using a multimode microplate reader (SuPerMax 3000FL, China). The percentage of cell viability was measured according to the following equation: where A t represents the mean absorbance of cells treated with SeNPs, and A c represents the mean absorbance of cells without SeNPs. Data are mean of triplicate experiments.
Wound healing assay. The scratch wound assay was used to measure the effect of SeNPs on the migration ability of HepG2 21,22 . Cells were cultured in 6-well plates. After 24 h, the cell monolayer was scratched with a 10 μL pipette tip to create a gap. After washing three times with serum-free medium, the gap was photographed to determine the wound baseline. Cells were incubated in FBS-free media with SeNPs solution at different concentrations. The wound gap size were evaluated at 24 h post-wounding. Image J was used for wound healing assay. The healing ratee was calculated according to the formula as following 21 : where A 0 represents the area of initial wound area, A f represents the remaining area of wound at 24 h. www.nature.com/scientificreports/ Statistical analyses. Statistical analyses were carried out by GraphPad Prism 9. One-way analysis of variance (ANOVA) followed was used to analyze the data. All data were presented as mean ± S.E.M. (n = 5). Significant differences were indicated with P value, *P < 0.05, **P < 0.001, ****P < 0.0001.
Ethical approval. This is an observational study. The "Preparation, characteristics and cytotoxicity of green synthesized selenium nanoparticles using Paenibacillus motobuensis LY5201 isolated from the local specialty food of longevity area" Research Ethics Committee has confirmed that no ethical approval is required. "

Results and discussion
Isolation and Identification of LY5201. Bama is "the hometown of longevity" in the world. Diet with adequate nutrition is one of the most important factors to longevity 23 . Trace elements play an important role in maintaining metabolic homeostasis in the elderly 24 . Since selenium rich area are related with longevity area, we tried to isolate the selenium-enriched strain from the local specialty food (Sauerkraut). In this study, LY5201 was isolated using LB plate supplemented with 5 mmol/L sodium selenite and exhibited the ability to reduce selenite to red Se 0 (Fig. 1a). As shown in Fig. 1b, 16S rRNA gene sequence and phylogenetic evolution analysis showed that LY5201 have a high degree of similarity (92%) with Paenibacillus motobuensis NR 043,153.1. It also exhibited the typical biochemical characteristics of P. motobuensis 25 as indicated in Table S1 (supporting data). The strain LY5201 was identified as P. motobuensis LY5210. It is Gram-negative, rod-shaped bacterium. In addition, this is the first study to prove that a P. motobuensis strain could reduce selenite to Se 0 and biosynthesize SeNPs. Recent studies have reported that hydroxyapatite nanoparticles could be synthesized by Acacia falcata leaf extract 26 and the fruit extract of Spondias pinnata could synthesize hematite nanoparticles 27 . It is considered that three steps were involved in the synthesis of nanoparticles by plants extracts: reduction of metal ions, nucleation and growth 28 . The secondary metabolites and biomolecules in plant extracts, such as carbohydrates and protein could reduce metal ions into stable nanoparticles and enhance their morphology. What's more, the reduction potential of the polyphenolic compounds in the extracts of plant are sufficient to reduce metal oxide to zero valence, which yields nanoparticles. Unlike the cell-free system in plant extracts systerm, synthesis of nanoparticles from microbes usually based on the whole cell. The mechanism of SeNPs synthesized from Se 0 varies among diverse microbial species including several metabolic pathways, enzymes and different proteins for the reduction process. It is considered that there are mainly three steps of SeNPs: (1) Transfer of selenite/selenate into the cells. Since elemental selenium scatters and absorbs radiation at 600 nm, which contributed to the increased absorbance 30 , the absorbance of the cell culture with selenite exceeded the value of control (0 g/L) and the growth rates at 1 g/L and 2 g/L selenite concentrations is higher than at 0.5 g/L selenite concentration. LY5201 has a higher level of selenite resistance than some other bacteria such as Azoarcus sp. CIB (8 mmol/L) 31 and Rhodopseudomonas palustris (8 mmol/L) 32 , which is similar to the selenite-tolerant strain, such as Alcaligenes faecalis (20.7 g/L) 13 .

Characterizations of P. motobuensis LY5201 and SeNPs. The purified SeNPs were characterized.
TEM was used to determine the location of SeNPs produced by P. motobuensis LY5201 (Fig. 3a). The naoparticles were located in the extracellular spaces. The particles were spherical and had homogenous size distribution. The average size of SeNPs was about 100 nm, similar to those found in Providencia rettgeri 16 and Lactobacillus casei 21 . More researches are needed to elucidate whether vesicular secretion is involved in the formation of SeNPs.
The XPS spectra showed that signals of C, O, N, S, P and Se were detected (Fig. 3b). The average particle size of SeNPs was obtained from the DLS analysis (Fig. 4a). The average poly dispersity index (PDI) was 0.227. Lower the PDI, the lesser the aggregation of particles. The main average size of the SeNPs was found at 130.4 ± 12.34 nm, which is larger than that in TEM analysis. It is possible that the size obtained from DLS does not only depend on the metallic core of SeNPs but also affected by the substances located on the surfaces such as bio-moieties and proteins 4 . The average size of SeNPs indicating that it can be used for biomedical applications such as biomaterial and bioactive drug deliver. As shown in Fig. 4b, SeNPs showed a negative zeta potential (-21.6 mv), indicating their stability in water. The presence of negatively charged functional groups on the surface of SeNPs were responsible for negative values of zeta potentials 33,34 .
The absorption spectra of SeNPs was shown in Fig. 4c. SeNPs exhibit a broad absorption peak at approximately 260 nm corresponds to previous study 7 , which confirms the formation of SeNPs. The UV spectra centered between 200 and 300 nm was due to the formation and surface plasmon vibration of SeNPs 35 . As shown in Fig. 4d, the FTIR spectra of the SeNPs showed absorption peaks at 3218 cm −1 , 1571 cm −1 , 1385 cm −1 , 1313 cm −1 , 1063 cm −1 , 770 cm −1 and 559 cm −1 . The peak at 3218 cm −1 corresponds to the O-H stretching, or to the N-H asymmetric stretch of proteins 4,33 . The peak at 1571 cm −1 corresponds to the amide II band of proteins 36  cell line at four doses: 0, 5, 10, 15 and 20 μg/mL. The viability of HepG2 cells were ranging from 83.9 ± 1.58% to 66.4% ± 6.94%. There are significant differences in cell viability among these groups (p < 0.05) (Fig. 5a). The scratch wound assay was carried out to evaluate the effect of SeNPs on the migration of HepG2 (Fig. 5b). Cell migration was examined via wound healing assay. SeNPs at different concentrations showed inhibitory effects on the migration of HepG2. The percentages of open wound were 31.52%, 21.71% and 1.86% in cells treated with SeNPs at 0, 10 and 20 μg/mL in a dose-dependent manner. SeNPs had been reported to have an anticancer activity on kidney, lung, liver and breast 12 . The anticancer mechanism of nanoparticles involves overproduction of ROS, depletion of mitochondrial membrane potential, and intracellular enzyme and protein interactions 37 . Oxidative stress can induce the cytotoxicity of nanoparticles to cancer cells, and DNA damage and cell mutation can lead to the damage of cancer cell development 38 . The results of our study are consistent with the published

Conclusion
The selection of the best biocatalysts to synthesize SeNPs in a fast and efficient manner is the most important factor in the SeNPs application. P. motobuensis LY5201 have the ability to synthesize extracellular SeNPs when growing with sodium selenite within 24 h. Organic-aqueous extraction is a successful method for collection of SeNPs. By this method, biomolecules modified-SeNPs was obtained. The SeNPs had cytotoxicity to HepG2 and inhibited the migration of HepG2. These results indicating that the SeNPs synthesized by P. motobuensis LY5201 may be used as a promising drug or biomaterial for hepatocellular carcinoma. P. motobuensis LY5201 could be used as a selenite bioconversion platform suitable for biological applications.  www.nature.com/scientificreports/

Data availability
The datasets generated during and/or analyzed during the current study are not publicly available as the data also forms part of an ongoing study, but are available from the corresponding author on reasonable request.